The Tonb System in Aeromonas Hydrophila NJ-35 Is Essential for Maca2b2 Efflux Pump-Mediated Macrolide Resistance

Dong et al. Vet Res (2021) 52:63 https://doi.org/10.1186/s13567-021-00934-w

RESEARCH ARTICLE Open Access The TonB system in Aeromonas hydrophila NJ‑35 is essential for MacA2B2 efux pump‑mediated macrolide resistance Yuhao Dong1, Qing Li2, Jinzhu Geng1, Qing Cao1, Dan Zhao1, Mingguo Jiang3, Shougang Li1, Chengping Lu1 and Yongjie Liu1*

Abstract The TonB system is generally considered as an energy transporting device for the absorption of nutrients. Our recent study showed that deletion of this system caused a signifcantly increased sensitivity of Aeromonas hydrophila to the macrolides erythromycin and roxithromycin, but had no efect on other classes of antibiotics. In this study, we found the sensitivity of ΔtonB123 to all macrolides tested revealed a 8- to 16-fold increase compared with the wild-type (WT) strain, but this increase was not related with iron deprivation caused by tonB123 deletion. Further study demonstrated that the deletion of tonB123 did not damage the integrity of the bacterial membrane but did hinder the function of macrolide efux. Compared with the WT strain, deletion of macA2B2, one of two ATP-binding cassette (ABC) types of the macrolide efux pump, enhanced the sensitivity to the same levels as those of ΔtonB123. Interestingly, the deletion of macA2B2 in the ΔtonB123 mutant did not cause further increase in sensitivity to macrolide resistance, indicating that the macrolide resistance aforded by the MacA2B2 pump was completely abrogated by tonB123 deletion. In addition, macA2B2 expression was not altered in the ΔtonB123 mutant, indicating that any infuence of TonB on MacA2B2-mediated macrolide resistance was at the pump activity level. In conclusion, inactivation of the TonB system signifcantly compromises the resistance of A. hydrophila to macrolides, and the mechanism of action is related to the function of MacA2B2-mediated macrolide efux.

Keywords: Aeromonas hydrophila, TonB system, Macrolides, MacA2B2 efux pump, Sensitivity

Introduction such as iron and vitamins that are present in the extracel- Gram-negative bacteria comprise most of the bacterial lular environment at very low concentrations [3]. Te sol- world. Diferent from Gram-positive bacteria, which utes that have a molecular mass less than 600 Da can pass only have a cytoplasmic membrane (CM), the cell enve- through OM porins by the concentration-gradient-driven lope of Gram-negative bacteria contains two membranes, passive difusion [4]. However, the uptake of nutrients, namely the CM and the outer membrane (OM) [1]. Te especially those existing at extremely low concentrations, OM protects Gram-negative bacteria from environmen- must occur by OM active transport. Due to the cell enve- tal hazards such as antibiotics and detergents [2]. Also, lope architecture, there is no electrochemical gradient Gram-negative bacteria require rare essential nutrients powering the active transport across the OM and no ATP in the periplasmic space, which means that OM trans- *Correspondence: [email protected] porters need to extract energy from the CM. Te energy 1 Joint International Research Laboratory of Animal Health and Food transfer is commonly carried out by trans-periplasmic Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, Jiangsu, China proteins, for example, the CM-anchored TonB complex Full list of author information is available at the end of the article [5]. TonB system, consisting of TonB, ExbB and ExbD

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proteins, couples the proton motive force (PMF) of the A. hydrophila and E. coli were routinely grown in CM to energize active transport across the OM by the Luria–Bertani (LB) medium at 28 °C and 37 °C, respec- TonB-dependent transporters (TBDTs) [6]. TonB pro- tively. When necessary, the medium was supplemented tein, the main component of this system, is CM-anchored with the following antibiotics: chloramphenicol (Cm), by a transmembrane helix, and stabilized by ExbB and 34 mg/L for E. coli; ampicillin (Amp), 100 mg/L for A. ExbD [7]. TonB system was thought to be only involved hydrophila. All reagents used in this study were sup- in nutrient import. In Escherichia coli, TonB system pro- plied by Sigma (St. Louis, MO, USA) unless otherwise vides energy to the OM protein BtuB for vitamin B 12 indicated. transport [3]. In Vibrio anguillarum, TonB1 system is responsible for heme and ferrichrome transport, while Minimum inhibitory concentration (MIC) assay TonB2 system is essential for the transport of endoge- A. hydrophila NJ-35 and its derivative tonB123 mutant nous and exogenous siderophores [8]. However, it is now grown to logarithmic phase were assayed for macrolide becoming clear that the uptake of nutrient is only one of sensitivity. MICs were determined by broth microdilu- the many potential functions of the TonB system. Indeed, tion, following the Clinical and Laboratory Standards in Myxococcus xanthus, TonB system is required for the Institute (CLSI) guidelines [14]. Briefy, cultured cells in 5 secretion of the protease PopC, which suggests that TonB the log phase were diluted to 2 × 10 cells/mL in fresh system not just participates in import processes, but also Mueller–Hinton broth (MHB). Te inoculum (100 μL) is involved in the secretion of intracellular proteins [9]. was added to each well of 96-well plates. Antibiotic was Aeromonas hydrophila is one of the most important then added to the frst wells and twofold dilutions were fsh pathogens causing haemorrhagic septicaemia. Also, performed. Plates were incubated at 28 °C for 18 h. Te this bacterium is associated with a variety of human MIC value was measured three times and the average of illnesses, such as sepsis, wound infections, and food- the measured values was determined as the MIC value borne gastroenteritis [10]. To date, antibiotics are still for the strain and the antibiotic. Macrolide antibiotics, the most efective way to fght this bacterial infection. including roxithromycin (ROX), erythromycin (ERY), However, the widespread use of antibiotics has led to the tilmicosin (TIL), tylosin (TYL), acetylspiramycin (ACE), emergence of antimicrobial resistance in A. hydrophila azithromycin (AZI), dirithromycin (DIR) and medemycin and bioaccumulation in host tissues, which seriously (MED) were purchased from Solarbio (Beijing, China). threatened human and public health. Some strains of A. To determine whether iron was involved in the efect hydrophila have been found to be resistant to macrolides, on the bacterial sensitivity to macrolides, MIC of each tetracyclines, sulfonamides, and quinolones [11, 12]. In macrolide was examined in MHB supplemented with response to this phenomenon, understanding of resist- 36 μM FeCl3 or 150 μM 2, 2-dipyridyl (DIP). ance mechanisms has become an urgent necessity for development of an efective therapeutic strategy against Cell membrane integrity this pathogen. To determine whether deletion of tonB123 resulted in Recently, three tonB genes in A. hydrophila NJ-35, damage to the permeability barrier of the bacterial cell namely tonB1, tonB2 and tonB3, were described to be membrane, cell membrane integrity was examined by involved in antibiotic resistance. A triple-deletion mutant determining the release of cytoplasmic constituents into of tonB123 showed a signifcantly increased sensitivity to the supernatant, such as nucleic acids and proteins [15]. the macrolide antibiotics erythromycin and roxithromy- Te bacteria were incubated at 28 °C for 6 h, and then cin, but had no efect on other classes of antibiotics [13]. immediately centrifuged (9000 g) for 5 min at 4 °C. Te In this study, we further demonstrated that the increased supernatant was flter-sterilized using 0.22-µm (pore- susceptibility of ΔtonB123 mutant to macrolides was due size) membrane. Te amount of nucleic acids released to the decrease in drug efux, and furthermore, associ- from the cytoplasm was determined by measuring the ated with the MacA2B2-mediated pump. optical density at 260 nm. Te concentration of proteins in the supernatant was determined using Protein Brad- ford Assay kit (Termo Fisher Scientifc, Waltham, USA). Materials and methods Bacterial strains and growth conditions Morphological observation Te bacterial strains and plasmids used in this study are Bacterial cell morphology was evaluated using scan- listed in Additional fle 1. A. hydrophila NJ-35 (accession ning electron microscope (SEM) and transmission elec- number CP006870), which belongs to the ST251 clonal tron microscope (TEM) [16]. For SEM examination, the group, was isolated from dead cultured cyprinoid fsh in dehydrated samples were treated thrice with 100% tert- the Jiangsu province of China in 2010. butanol and dried with a freeze dryer for 2 h. Te samples Dong et al. Vet Res (2021) 52:63 Page 3 of 10

were placed on stubs and coated with gold flm by sputter chromosomal DNA of A. hydrophila NJ-35, and then coating and viewed using a FEI Quanta FEG250 scanning ligated in-frame using fusion PCR. Te fusion frag- electron microscope. For TEM examination, the dehy- ment was cloned into the pYAK1 suicide plasmid and drated samples were infltrated, embedded in araldite then chemically transformed into E. coli SM10 compe- and processed to a trapezoid shape having a surface tent cells. Te donor strain E. coli SM10-pYAK1 and the area of less than 0.2 mm × 0.2 mm. Ultramicrotomy was recipient strain A. hydrophila were mixed at a ratio of performed on the embedded material to obtain a thick- 2:1 (vol/vol) in medium, spotted on a nylon flter on an ness of 50–90 nm. Subsequently, the thin sections were LB plate and conjugated for 12 h at 28 °C. Te bacteria mounted onto 300 mesh copper grids, stained with alco- were washed from the flter and grown on LB plates con- holic uranyl acetate and alkaline lead citrate, and washed taining Amp and Cm. Te positive colonies were verifed with distilled water. Te samples were observed on a by PCR and then inoculated in LB broth supplemented Hitachi 600 transmission electron microscope. with 20% sucrose to induce a second crossover event. Te suspected mutants were verifed by PCR. Using the Drug accumulation assay same approach, additional deletion mutants were also To determine the infuence of TonB system on macrolide constructed. efux, the intracellular drug accumulation was exam- Te corresponding complemented strains of the ined with the WT and ΔtonB123 strains. A single col- mutants were constructed with pMMB207. Te target ony of each strain was cultured in LB medium at 28 °C gene was isolated by PCR amplifcation and then ligated for 6 h, after which the cells were transferred to 100 mL into pMMB207. Te verifed complementation vector LB medium and cultured to the logarithmic-phase was transferred into E. coli SM10 by chemical transfor- (OD600 = 0.6). Cells were pelleted by centrifugation at mation and then transformed into the mutant strain by 4000 g for 10 min, and resuspended in PBS to a turbidity conjugation. Te transconjugants were selected on LB at 600 nm of about 3.0. Te cells were then incubated for agar containing Amp and Cm and further confrmed by 15 min at 28 °C, and tilmicosin was added to reach the PCR. fnal concentration of 10 mg/L. 1 mL aliquots of culture were removed from the tube every 5 min. Ten cells were Quantitative reverse transcription PCR (qRT‑PCR) harvested by centrifugation at 800 g for 10 min. Te pel- Total RNA was extracted from cultures in the exponen- let was washed three times with PBS, followed by soni- tial growth phase with an E.Z.N.A. bacterial RNA isola- cation on ice, then centrifuged at 10 000 g for 10 min to tion kit (Omega, USA). Te RNA was reverse transcribed remove cellular debris. Te intracellular concentration to cDNA using HiScript II QRT Supermix (Vazyme, of tilmicosin was analysed by high-performance liquid China). Te mRNA transcription levels were examined chromatography coupled with tandem mass spectrom- using a One Step qRT-PCR SYBR Green kit (Vazyme etry (HPLC–MS/MS) [17]. Biotech) in an ABI PRISM 7300 Fast Real-time PCR machine. All qRT-PCR operations were performed in Efect of efux pump inhibitors on the sensitivity of A. triplicate. Te housekeeping gene recA was chosen as hydrophila to macrolides an internal control for qRT-PCR, and the fold-change of To assess the contribution of the efux pump to TonB- mRNA expression levels was calculated according to the dependent macrolide efux, MIC levels were deter- 2−∆∆CT method [18]. Te primer pairs are shown in Addi- mined using broth microdilution assay in the absence tional fle 2. or presence of 3 mg/L carbonyl cyanide m-chlorophenylhydrazone (CCCP), 50 mg/L phenylalanine-arginine Statistical analyses β-naphthylamide (PAβN) or 10 mg/L sodium orthovana- Data were analysed using SPSS16.0 software (SPSS Inc., date (SOV). Te fnal concentrations of the pump inhibi- Chicago, IL, USA). Multiple comparisons were per- tors were selected based on a preliminary sighting study, formed using a Student t test and analysis of variance in which the highest concentration of efux pump inhibi- (ANOVA) followed by Bonferroni’s post hoc-test. P-val- tor that does not afect bacterial growth was determined. ues < 0.05 were considered to be statistically signifcant.

Inactivation of the target gene Results Gene mutants were constructed by homologous recom- Inactivation of tonB123 enhances A. hydrophila sensitivity bination using the suicide plasmid pYAK1 as previously to macrolide antibiotics described [13]. Te primer pairs are shown in Additional To confrm the role of TonB system in macrolide sensitiv- fle 2. Briefy, the upstream and downstream fanking ity of A. hydrophila, we tested the sensitivity of the WT regions of the target gene were PCR amplifed from the and ΔtonB123 strains to common macrolides, including Dong et al. Vet Res (2021) 52:63 Page 4 of 10

erythromycin, roxithromycin, dirithromycin, azithromy- this fnding, we determined the MIC of each macrolide in cin, acetylspiramycin, tilmicosin, tylosin and medemycin. the presence of the iron chelator DIP. Te results showed Te MIC data showed that the sensitivity of ΔtonB123 that iron deprivation also had no efect on the macrolide strain to all above macrolides increased 8- to 16-fold sensitivity of the WT or ΔtonB123 strains (Figure 3). compared with the WT strain (Table 1). Tis defect could be partially complemented by any of the three tonB Inactivation of tonB123 hinders macrolide efux function genes. Tese results indicated an important role of TonB To investigate whether the efect of TonB system on system in macrolide resistance. macrolide resistance is related to the efux pump activity, we determined the MIC of macrolides in presence of Inactivation of tonB123 does not damage the bacterial the efux pump inhibitor PAβN. Our data showed that membrane PAβN eliminated the diference of macrolide resistance To determine whether inactivation of the TonB system between the WT strain and ΔtonB123 mutant (Figure 4). disrupted the cell membrane integrity, we monitored the Tis fnding suggests that efux pumps might be involved leakage of cell constituents through the bacterial mem- in mediating macrolide resistance in the ΔtonB123 brane. As shown in Figure 1A, the amount of nucleic mutant. To further assess whether the increased sensitiv- acids released in ΔtonB123 was not diferent from that of ity of ΔtonB123 results from the disruption in function the WT strain. Similarly, the WT and ΔtonB123 strains of antibiotic efux, a tilmicosin accumulation assay was had the same amount of protein leakage (Figure 1B). In performed with the WT and ΔtonB123 strains. As dem- addition, as demonstrated by SEM and TEM, both the onstrated in Figure 5, the ΔtonB123 mutant accumulated WT and ΔtonB123 strains displayed intact cell mem- more tilmicosin compared to the WT strain (P < 0.001). brane and smooth surface, with no observable diference Tese data indicate that deletion of TonB system might (Figure 2). somehow infuence the behaviour of A. hydrophila in macrolide efux. Iron availability does not infuence the sensitivity of A. hydrophila to macrolides ATP‑binding cassette (ABC) family efux is involved To investigate if iron deprivation could infuence mac- in TonB‑dependent macrolide resistance rolide susceptibility of A. hydrophila, we determined To explore which kind of pump was involved in TonB- the MIC of each macrolide in MH broth supplemented dependent macrolide efux, we determined the MICs with an excess of iron (36 μM FeCl3). Our data revealed of macrolides against WT and ΔtonB123 strains in the that the sensitivity of ΔtonB123 mutant to macrolides presence of CCCP, an ionophore that disrupts PMF. increased by 8- to 16-fold compared to that of the WT Te results showed that addition of CCCP caused sig- strain, regardless of iron presence (Figure 3). Te results nifcantly increased susceptibility of both the WT and suggested that iron surplus could not restore the mac- ΔtonB123 strains to all macrolides, but a signifcant dif- rolide susceptibility in the ΔtonB123 mutant. To confrm ference in macrolide sensitivity between the two strains

Table 1 Infuence of tonB123 deletion on the susceptibility of A. hydrophila strains to macrolide Strain MIC (mg/L) ROX ERY TIL TYL ACE AZI DIR MED

WT 32 16 64 128 256 1 128 128 ΔtonB123 2 1 4 16 16 0.125 16 8 ΔtonB123 ptonB1 16 8 32 64 64 1 64 128 + ΔtonB123 ptonB2 16 8 32 64 64 1 64 128 + ΔtonB123 ptonB3 8 4 16 32 32 0.25 64 64 + ΔmacA1B1 32 16 64 128 256 1 128 128

ΔtonB123ΔmacA1B1 2 1 4 16 16 0.125 16 8

ΔmacA2B2 2 1 4 16 16 0.125 16 8

ΔtonB123ΔmacA2B2 2 1 4 16 16 0.125 16 8 ΔmacA B pmacA B 32 16 64 128 256 1 128 128 2 2 + 2 2 ΔtonB123ΔmacA B pmacA B 2 1 4 16 16 0.125 16 8 2 2 + 2 2 ROX: roxithromycin, ERY: erythromycin, TIL: tilmicosin, TYL: tylosin, ACE: acetylspiramycin, AZI: azithromycin, DIR: dirithromycin, MED: medemycin. Dong et al. Vet Res (2021) 52:63 Page 5 of 10

was mediated by these two pumps, we deleted the macA1B1 and macA2B2 loci, respectively, in the WT and ΔtonB123 background. As shown in Table 1, inactivation of macA1B1 had no efect on macrolide susceptibility of the two strains, but deletion of macA2B2 in the WT strain enhanced the susceptibility to the same levels as those of the ΔtonB123 mutant. Notably, deletion of macA2B2 in the ΔtonB123 mutant did not further enhance the improved bacterial susceptibility to macrolides. Tis fnding indicated that macrolide resistance aforded by the MacA2B2 pump was completely abrogated by deletion of tonB123. Further, we evaluated the mRNA transcription levels of two components of the macA2B2 locus in the WT and ΔtonB123 strains by qRT-PCR. No signifcant dif- ference was observed between the two strains (Fig- ure 6), indicating that the efect of the TonB system on MacA2B2-mediated macrolide resistance was not related to the gene expression level of the efux pump.

Discussion In agreement with our recent investigations, this study further demonstrated the involvement of the TonB system in macrolide resistance of A. hydrophila. Te TonB system in Pseudomonas aeruginosa has been reported to be related to resistance to various classes of antibiotics, such as quinolones, macrolides, penicillins, and β-lactams [22]. In Neisseria gonorrhoeae, the TonB system was required for antimicrobial hydrophobic agents, Figure 1 Leakage of nucleic acids (A) and proteins (B) from the as tonB-exbB-exbD deletion mutant showed increased WT and ΔtonB123 mutant. Nucleic acids leakage was assessed by sensitivity to erythromycin, polymyxin B and detergent measuring the absorbance of the supernatants of bacterial cultures at Triton X-100 [23]. Diferent from the above two reports, OD260. The protein concentration in supernatants was determined by the Bradford’s method. Data represent the means SEM from three our previous fndings for A. hydrophila demonstrated ± independent experiments. “ns” signifes not statistically signifcant. that the TonB system was only involved in tolerance to macrolides. Te TonB system is an energy transduction complex that consists of the ExbB, ExbD and TonB proteins was still present (Figure 4). However, when the ABC fam- which delivers energy from the CM to the OM for nutri- ily efux was restrained by the ATPase inhibitor SOV, the ent transport [24]. Our previous study indicated that the diference in sensitivity to macrolides between WT and TonB system in A. hydrophila NJ-35 was involved in iron ΔtonB123 disappeared completely (Figure 4). Tese data transport [13]. It is well known that intracellular iron suggested that the TonB system might impact ABC fam- homeostasis plays an important role in bacterial antibi- ily pump-mediated macrolide efux. otic resistance. Iron deprivation is known to be able to impair the activity of drug efux pump in Mycobacterium tuberculosis [25]. In this study, however, addition of an TonB is essential for the function of the MacA2B2 efux iron overload did not change the sensitivity of the wild- pump type A. hydrophila or its tonB123 mutant. Tis fnding Te macrolide-specifc ABC-type efux pump MacAB indicates that the enhanced efect of tonB123 deletion on has been identifed in diverse Gram-negative bacte- macrolides susceptibility in A. hydrophila was independ- ria [19–21]. In the genome of A. hydrophila NJ-35, two ent of its infuence on iron availability. putative open reading frame (ORF) clusters, MacA 1B1 Tere is a great variety of resistance mechanisms and MacA2B2, were retrieved. To determine whether observed in bacteria. Of them, the OM barrier and efux the efect of the TonB system on macrolide resistance pumps are the two most common resistance mechanisms Dong et al. Vet Res (2021) 52:63 Page 6 of 10

Figure 2 Bacterial cell morphology of the WT and ΔtonB123 mutant. A SEM images of the WT and ΔtonB123 mutant. Scale bar 1 μm. B TEM = images of the WT and ΔtonB123 mutant. Scale bar 500 nm. At least three random felds were observed and analyzed, from three independent = experiments.

[26]. Te former limits the rate of antibiotic uptake into pump for macrolide extrusion was compromised in the the cell, while the latter can expel drug into the exter- ΔtonB123 mutant. Te diference between the WT and nal surrounding environment [22]. Te TonB system ΔtonB123 strains in macrolide sensitivity disappeared in has been demonstrated to be necessary for maintaining the presence of a broad-spectrum efux pump inhibitor membrane integrity in Pseudomonas putida, and its def- PAβN, suggesting that the TonB system is associated with ciency leads to increased membrane permeability [27]. efux of macrolides. Bacterial efux systems generally In this study, however, inactivation of this system did not fall into fve classes, the major facilitator (MF) superfam- afect membrane permeability. Moreover, increased sen- ily, the resistance-nodulation-division (RND) family, the sitivity observed in the ΔtonB123 was shown to be drug small multidrug resistance (SMR) family, the multidrug specifc. We also tested whether functioning of the efux and toxic compound extrusion (MATE) family and the Dong et al. Vet Res (2021) 52:63 Page 7 of 10

Figure 3 Infuence of iron on macrolide resistance of the WT and ΔtonB123 mutant. The MICs were determined in MH broth containing

36 μM FeCl3 (iron-supplemented) or 150 μM DIP (iron-limited). The data represent three independent biological replicates.

Figure 4 Efect of efux pump on TonB-dependent macrolide efux in the WT and ΔtonB123 mutant. The MICs were determined in the absence or presence of 3 mg/L CCCP, 50 mg/L PAβN or 10 mg/L SOV. The data represent three independent biological replicates.

ABC family [28]. Except for the ABC transporters, which However, our study shows for the frst time that the efect use the ATP as an energy source to transport the drugs of the TonB system in A. hydrophila macrolide resistance across the membrane, the others described are PMF- was mediated by MacA 2B2 efux pump, which has been dependent multidrug efux pumps [29]. Several previ- reported as an ABC family pump. ous studies have shown that the TonB system participates In Gram-negative bacteria, MacAB efux pumps usu- in the efux of antibiotics through RND efux pumps, ally cooperate with outer membrane channel TolC to for example, MexAB-OprM pump in P. aeruginosa [22] fulfll the efux function [30]. MacAB forms a tripar- and MtrC-MtrD-MtrE pump in N. gonorrhoeae [23]. tite channel with TolC to drive the efux of macrolides Dong et al. Vet Res (2021) 52:63 Page 8 of 10

number 1NQH) [33, 34]. Terefore, TolC is unlikely to be a TonB-dependent gated channel. A common fea- ture in action mechanisms of TonB system is that TonB behaves as a regulating protein that infuences the con- formation of outer membrane proteins. Terefore, we speculate that TonB may be indirectly involved in the allosteric mechanism of TolC by protein–protein interactions. However, we cannot rule out the possibility that TonB defciency may interfere with the activation of the pump components located in the cytoplasmic membrane, making the pump nonfunctional. Neverthe- less, these questions are actively being studied in our group and remain exciting for the future. Figure 5 Accumulation of tilmicosin in the WT and ΔtonB123 mutant. The intracellular concentration of tilmicosin was analysed Taken together with our recent report, these results by HPLC–MS/MS. Data represent the means SEM from three support the notion that TonB system plays an important ± independent experiments. ***P < 0.001. role in macrolide resistance of A. hydrophila. Although the exact mechanism warrants continued study, the TonB system is clearly involved in the resistance action by inhibiting the function of MacA 2B2-mediated macrolide efux. Further elucidation of the mechanism of action will undoubtedly contribute to the development of new antimicrobial agents.

Abbreviations WT: Wild-type; ABC: ATP-binding cassette; CM: Cytoplasmic membrane; OM: Outer membrane; PMF: Proton motive force; TBDTs: TonB-dependent transporters; LB: Luria–Bertani; Cm: Chloramphenicol; Amp: Ampicillin; MIC: Minimum inhibitory concentration; CLSI: Clinical and Laboratory Standards Institute; MHB: Mueller Hinton broth; DIP: 2,2-Dipyridyl; SEM: Scanning electron microscope; TEM: Transmission electron microscope; OD: Optical density; HPLC–MS/MS: High-performance liquid chromatography coupled with tandem mass spectrometry; CCCP: Carbonyl cyanide m-chlorophenylhydrazone; PAβN: Phenylalanine-arginine β-naphthylamide; SOV: Sodium orthovanadate; PCR: Polymerase chain reaction; ORF: Open reading frame; MF: Major facilitator; RND: Resistance-nodulation-division; SMR: Small multidrug resistance; Figure 6 The mRNA expression levels of two components of MATE: Multidrug and toxic compound extrusion; ATP: Adenosine triphosphate; the macA2B2 locus in the WT and ΔtonB123 mutant. RNA was ROX: Roxithromycin; ERY: Erythromycin; TIL: Tilmicosin; TYL: Tylosin; ACE: Ace- isolated from the log-phase bacteria that had been cultured in LB tylspiramycin; AZI: Azithromycin; DIR: Dirithromycin; MED: Medemycin. broth. The recA gene was chosen as the internal control. The results are expressed as n-fold increase with respect to the control. Data represent the means SEM from three independent experiments. “ns” Supplementary Information ± signifes not statistically signifcant. The online version contains supplementary material available at https://doi. org/10.1186/s13567-021-00934-w.

Additional fle 1. Bacterial strains and plasmids used in this study. out of the bacterial cells [31]. Given that the function Additional fle 2. Primers used in this study. of the TonB system is to energize the TBDTs in the OM Additional fle 3. The 3D structure of TolC (A) and BtuB (B). The 3D for the transport of nutrients, it is tempting to specu- structure of TolC (U876_06560) was predicted used I-TASSER online server. late that TonB proteins may transfer PMF from the Three TolC monomers assemble to form a continuous conduit containing a 12-stranded β-barrel and a α-helical barrel. The monomers are individu- cytoplasmic membrane to TolC to allow for antibiotic ally coloured. In the single protomer, the β-barrel domain is in blue, the efux. In this study, however, the protein domain pre- a-helical barrel domain is in orange. BtuB is the E. coli TonB-dependent dicted used I-TASSER online server [32] showed that vitamin B12 transporter. In BtuB, TonB box is in red, luminal domain is in purple and the β-barrel is in yellow. Protein Data Bank accession number TolC did not contain a beta-barrel or lumen-occlud- 1NQH. ing cork domain with an essential sequence called the TonB box (Additional fle 3), which was the typical Acknowledgements structure of TBDT BtuB (Protein Data Bank accession We thank Maoda Pang for his valuable review and comments on early drafts of this article. Dong et al. Vet Res (2021) 52:63 Page 9 of 10

Authors’ contributions comparative genomic and functional analyses of virulence genes. MBio YD and QL carried out most of the experiments described in the manuscript 4:e00064-00013 and wrote the article; QL, JG, QC, DZ, MJ and SL participated in the design of 11. Skwor T, Stringer S, Haggerty J, Johnson J, Duhr S, Johnson M, Seckinger the study and performed the statistical analysis. YL provided expertise and M, Stemme M (2020) Prevalence of potentially pathogenic antibiotic- conceived the study. All authors read and approved the fnal manuscript. resistant Aeromonas spp. in treated urban wastewater efuents versus recipient riverine populations: a 3-year comparative study. Appl Environ Funding Microbiol 86:e02053-e2119 This study was funded by National Natural Science Foundation of China 12. 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